Human Diseases Caused by Mutation in Splicing Signals

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Since alternative splicing plays such an important role in gene expression, it is not surprising that an increasing number of diseases are caused by abnormal splicing patterns (Stoilov et al. 2002; Faustino and Cooper 2003; Garcia-Blanco et al. 2004; Fig. 1B). There is a positive correlation between the number of splice sites and the likelihood of a gene causing a disease, suggesting that many mutations that cause diseases may actually disrupt the splicing pattern of a gene (Lopez-Bigas et al. 2005). The disease-causing mechanism can be subdivided into changes in cis- and trans-factors. Changes in cis-factors are caused by mutations in splice sites, silencer and enhancer sequences, and through generation of novel binding sites in triplet repeat extensions. Alterations in trans-acting factors are frequently observed in tumor development, where the concentration and ratio of individual trans-acting factors change. Mutations can be seen as new sources for alternative splicing regulation. For example, the alternative splicing patterns of different histocompatibility leukocyte antigens (HLA) are regulated by allele-specific mutations in the branchpoint sequences. Since the variability of HLAs are the basis of the adaptive immune response, these mutations strengthen the immunity by enlarging the number of potential HLA molecules (Kralovicova et al. 2004).

Mutation of Cis-acting Elements

Mutations of cis-acting elements can be classified according to their location and action. Type I mutations occur in the splice sites and destroy exon usage, type II mutations create novel splice sites that cause inclusion of a novel exon, type III and IV mutations occur in exons or introns, respectively, and affect exon usage. Type I and II mutations are the simplest mutation to be recognized. About 10% of the mutations stored in the Human Gene mutation database affect splice sites. They have been compiled in that (Stenson et al. 2003) and in specialized databases (Nakai and Sakamoto 1994).

Although bioinformatics resources such as the ESE finder (Cartegni et al. 2003), or the RNA workbench (Thanaraj et al. 2004) help to predict type III and IV mutations, the theoretical models often do not fit the experimental findings (Pagani et al. 2003a). However, the increase of genotype screening in human diseases has identified numerous exonic and intronic variations. Their association with a disease phenotype is often unclear since apparently benign polymorphism, such as codon third position variations or conservative amino acid replacement, are difficult to assess. A list of well-studied mutations in splicing regulatory elements is given in Table 1 and is maintained at the alternative splicing database web site (http://www.ebi.ac.uk/asd/).

Examples of Diseases

As examples, we discuss two well-studied pathologies: cystic fibrosis and spinal muscular atrophy. Cystic fibrosis is a recessive disease caused by loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) gene occurring with an incidence of 1:3,500. The CFTR gene encodes a cAMP-regulated chloride channel that controls the hydration of mucus. Currently, 1,388 mutations of CFTR have been described, 185 of which are splicing mutations. Twenty of these splicing mutations are located in exons, the rest in introns (http://www.genet.sickkids.on.ca/cftr/), which roughly reflects the exon/intron composition of the gene. Mutations changing exons 9 and 12 usage have been studied in detail. Both exons are alternatively spliced in healthy individuals and the ratio of exon inclusion varies between individuals (Hull et al. 1994), which could be attributed to variable concentrations of trans-acting factors between them. Complete skipping of these exons is caused by several splice-site mutations. These mutations result in the classical clinical picture of cystic fibrosis that shows chronic respiratory and digestive problems, and affects the lower respiratory tracts, pancreas, biliary system, male genitalia, intestine, and sweat glands. In contrast, type III and IV mutations change the ratio of exon inclusion and cause non-classical forms of cystic fibrosis that affect only a subgroup of organs or appear later. A detailed analysis of the mutations showed that they are part of a larger regulatory element, the composite exonic regulatory element of splicing (CERES). CERES contains multiple overlapping silencing and enhancing elements that work only in the particular CERES context and cannot be moved into heterologous sequence contexts. Several neutral polymorphisms in CERES can influence splicing and therefore contribute to the disease. Finally, the isoform ratio evoked by CERES mutation was depending on the cell type, which would explain why the mutations affect only a few organs (Pagani et al., 2003a; Pagani et al., 2003b). Thus, mutations affecting alternative splicing contribute to a very heterogeneous clinical phenotype that makes genotype-phenotype correlation difficult.

Spinal muscular atrophy is a neurodegenerative disorder with progressive paralysis caused by the loss of alpha motor neurons in the spinal cord. The incidence is 1:6,000 for live births and the carrier frequency is 1 in 40, making SMA the second most common autosomal recessive disorder and the most frequent genetic cause of infantile death. SMA is caused by the loss of the SMN1 gene that encodes the SMN protein, which regulates

Table 1. Examples of enhancer mutations involved in human diseases. The table lists examples of mutations in regulatory motifs that cause aberrant splicing. The list is updated at the alternative splicing database website (www.ebi.ac.uk/asd/). Large letters indicate exonic mutations, small letters indicate intronic mutations. The top line of each sequence indicates wild type, the lower line the mutant

Disease

Gene

Mutation

Reference

FTDP-17

FTDP-17

FTDP-17

FTDP-17

Thrombasthenia of Glanzmann and Naegeli tau tau tau tau

Integrin GPIIIA

ATTAATAAGAAG

ATTAAGAAGAAG

AAG del at 16 of ExonlO (280 K)

ATTAATAAGAAGCTG

ATTAAT AAGCTG

CTGGATCTTAGCAAC

CTGGATCTCAGCAAC

G>A at pos. 92 of ExonlO (S305 N) improves the splice site

GGCAGTGTGA

GGCAATGTGA

ACGGTGAGgt ACAGTGAGgt

Iijima et al. (1999)

Menkes disease

GATCTTCTGGA GATCT GGAT

Metachromatic leukodystrophy Immunodeficiency

Cerebrotendinous xanthomatosis Marfan syndrome

Arylsulfatase A

TNFRSF5, tumor-necrosis factor receptor superfamily, member 5 (CD40); CYP27A1

Fibrillin-1

Acute intermittent porphyria

Hereditary tyrosinemia

Leigh's encephalomyelo-pathy

Immunodeficiency

Porphobilinogen deaminase

Fumarylacetoacetate hydrolase Pyruvate dehydrogenase El alpha

Adenosine deaminase

CAGACGAGGTC CAGACAAGGTC CTACAGGG CTACTGGG

CCTATGGGCCGTT

CCTATGTGCCGTT

GGGATCATCGTGGGA

GGGATCATTGTGGGA

GTGATTCGCGTGGGT

GTGATTCGGGTGGGT

CTTATGAACGACTGG

CTTATGAATGACTGG

GGGCGCTGG GGGCACTGG

GGGGAGCGAGACTTC GGGGAGTGAGACTTC

Hasegawa et al. (1994) Ferrari et al. (2001)

Llewellyn et al. (1996)

Ploos van Amstel et al. (1996)

De Meirleir et al. (1994)

Santisteban et al. (1995)

(Continued)

Table 1. Examples of enhancer mutations involved in human diseases. The table lists examples of mutations in regulatory motifs that cause aberrant splicing. The list is updated at the alternative splicing database website (www.ebi.ac.uk/asd/). Large letters indicate exonic mutations, small letters indicate intronic mutations. The top line of each sequence indicates wild type, the lower line the mutant—(Cont'd)

Table 1. Examples of enhancer mutations involved in human diseases. The table lists examples of mutations in regulatory motifs that cause aberrant splicing. The list is updated at the alternative splicing database website (www.ebi.ac.uk/asd/). Large letters indicate exonic mutations, small letters indicate intronic mutations. The top line of each sequence indicates wild type, the lower line the mutant—(Cont'd)

Disease

Gene

Mutation

Reference

2-methylbutyryl-coa

Short/branched-chain

GAGTGGATGGGGG

Matern et al. (2003)

dehydrogenase deficiency/

acyl-CoA

GAGTGGGTGGGGG

short/branched-chain

dehydrogenase

acyl-coa dehydrogenase

(SBCAD)

Homocystinuria

Methionine synthase

TCAGCCTGAGAGGA

Zavadakova et al. (2002);

TCAGCCCGAGAGGA

Zavadakova et al. (2005)

Bardet-Biedl

MGC1203

GGCCTTCG

Badano et al. (2006)

Syndrome

GGCCTTTG

snRNP assembly. Humans posses an almost identical gene, SMN2 that was generated through a recent duplication. Although both genes are almost identical in sequence, due to a translationally silent C>T change at position 6 in exon 7, they have different splicing patterns and exon 7 is predominantly excluded in SMN2. This exon-skipping event generates a truncated, less stable and probably nonfunctional protein. Therefore, SMN2 cannot compensate the loss of SMN1. The SMN protein functions in the assembly of snRNPs and is absent from all cells in SMA patients. However, this protein deficiency becomes only apparent in motor neurons that eventually die. The loss of the motor neurons causes SMA. The disease can manifest in four phenotypes (type I to IV) that differ in onset and severity. The phenotypes correlate roughly with the number of SMN2 copies in the genome, most likely because more SMN2 copies produce more SMN protein. Since stimulation of SMN2 exon 7 usage would increase SMN protein levels and potentially cure the disease, work has concentrated on understanding the regulation of exon 7. As for CFTR exon 9 and 12, multiple factors determine the regulation, including a suboptimal polypyrimidine tract (Singh et al. 2004c), a central tra2-beta1-dependent enhancer (Hofmann et al. 2000) and the sequence around the C>T change at position 6 that can either bind to SF2/ASF or hnRNPA1 (Cartegni and Krainer 2002; Kashima and Manley 2003). Recent large scale mutagenesis studies indicate that again a composite regulatory exonic element termed EXINCT (extended inhibitory context) is responsible for the regulation of exon 7 inclusion (Singh et al. 2004a; Singh et al. 2004b).

These two examples illustrate some of the general principles of diseases caused by misregulated splicing: mutations in splicing regulatory sequences can be hard to detect and translationally silent point mutations or intronic mutations can have drastic effects. The effect of the identical mutation on splice site selection can vary between cell types, which can cause specific, sometimes atypical, phenotypes. Identical mutations show also different penetrance when different individuals are analyzed, suggesting that alternative splicing could be a genetic modifier (Nissim-Rafinia and Kerem 2002).

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  • Samsa
    What disorders are caused by aberrant gene splicing?
    11 months ago
  • semret
    Does mutation of exon vary in infection?
    9 months ago

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